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The extended interface: measuring non-local effects in biomolecular interactions
The extended interface: measuring non-local effects in biomolecular interactions John E Ladbury1 and Mark A Williams2 Improvements in the sensitivity and availability of biophysical techniques for the detection of the formation of complexes in solution are revealing that the effects of binding are not restricted to the direct contacts between the biomolecules or even to a localised site. Rather, information about the binding event is transmitted throughout the biomolecules and the surrounding solution through changes in the hydrogen bonding, hydration and electrostatic field as the complex is formed. Calorimetric, volumetric and NMR methods are beginning to provide a quantitative view of the nature and thermodynamic consequences of this extended interface, and the resulting data pose a major challenge for computational models of binding. Addresses Department of Biochemistry and Molecular Biology, and Institute for Structural Molecular Biology, University College London, Gower Street, London WC1E 6BT, UK 1 e-mail: [email protected] 2 e-mail: [email protected]
and entropy (DS) resulting from the binding process [1]. However, the specific attribution of thermodynamic parameters to the formation/breaking of particular local non-covalent interactions, to conformational or dynamic change, or to solvent reorganisation is not easy to achieve. Even knowledge of the crystal structures of both the free biomolecules and the complex is not enough to allow us to explain the binding thermodynamics. The true extent of conformational change and dynamics can be hidden by crystal packing effects, and mobile parts of the structure, water molecules and salt, are usually indistinguishable or undetectable in smeared or low electron density. Consequently, for a complete understanding of the binding event, we need extensive solution-state experimentation. In this review, we highlight instruments and methodologies that can be employed to deconvolve the non-local structural and thermodynamic effects associated with binding, and show that their application is leading to a fundamental change in the way we think about biomolecular interactions.
Current Opinion in Structural Biology 2004, 14:562–569 This review comes from a themed issue on Biophysical methods Edited by Arthur G Palmer III and Randy J Read Available online 15th September 2004 0959-440X/$ – see front matter # 2004 Elsevier Ltd. All rights reserved. DOI 10.1016/j.sbi.2004.08.001
Abbreviations ITC isothermal titration calorimetry PwTBP Pyrococcus woesei TATA-binding protein
Introduction The idea that biomolecular interactions can be understood simply in terms of the coming together of two rigid bodies to create several specific interactions at a welldefined binding site is progressively being eroded by detailed studies of equilibrium binding processes in solution. There are an increased number of recent reports of binding events that are accompanied by significant structural and dynamic changes of the interacting partners, or perturbation of the surrounding solvent. Often, these changes are distant from the site of intermolecular contact itself, but their contribution to the thermodynamics of binding can be considerable. Consequently, their characterisation is essential to a complete understanding of biological mechanism. Calorimetric methods can determine the total change in free energy (DG), enthalpy (DH) Current Opinion in Structural Biology 2004, 14:562–569
Global thermodynamics of binding Although it has been possible for many years to study the thermodynamics of a binding event indirectly via spectroscopic observation of the bound fraction during a titration, instrumentation that allows a direct and complete thermodynamic characterisation of biomolecular interactions has only recently become commercially available. Isothermal titration calorimetry (ITC) directly determines the total DH of binding and the equilibrium binding constant, KB, is simultaneously determined by using the measured heat as a probe of the degree of binding as two biomolecules are titrated against one another. A complete thermodynamic characterisation of an interaction can be achieved using the relationships: KB ¼ eDG=RT
(1)
DH ¼ DG þ T DS
(2) 1
1
where R is the gas constant (8.3 J.mol .K ) and T is the experimental temperature. Repetition of the experiment at several temperatures (and constant pressure) yields the change in heat capacity (DCp) that accompanies binding: DCp ¼
@DH @T
(3)
There are already several reviews detailing instrument design, usage and applications of the basic ITC technique [1–3]; here, we focus on methods that give insight into the contribution of the ionic environment, protein conformational change and water molecules to binding www.sciencedirect.com
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thermodynamics. In order to deconvolve specific contributions, a series of experiments is performed in which a single physical parameter related to the contribution or contributions of interest is systematically varied.
The counterion cloud The contribution of the ionic environment can be experimentally assessed by thermodynamic characterisation at several distinct salt concentrations. Intracellular salt concentrations vary widely, from approximately 200 mM in human tissue to more than 2 M in some halophilic archaea, and their effects can be profound, particularly for oppositely charged binding partners (e.g. the KB of the purine repressor protein for its operator DNA sequence falls by four orders of magnitude as the salt concentration is changed only twofold from 200 mM to 400 mM [4]). The partial cancellation of the electrostatic fields of the protein and DNA as they form the complex radically reduces the range of their effect on the surrounding solution, and the field close to the interface is extensively modified through the focusing effect of the low dielectric biomolecules [5]. Counterions in solution redistribute in response to these changes in order to minimise the free energy of the solute–solvent system. For protein–DNA complexes, an empirical relationship has been established between the salt concentration and the binding constant (derived from [6]): log KB ¼ log KB;ref Alog½Salt þ 0:016B½Salt
(4)
where the constants A and B are interpreted, respectively, as the number of ions and the number of water molecules released to bulk from the negative polyelectrolyte DNA and largely positive protein surfaces that are occluded upon binding [7]. KB,ref is the hypothetical binding constant at 1 M salt and B = 0. The thermodynamic contribution to the salt dependence from the release of water molecules is small below 1 M salt and is a bulk solution property that is not affected by the electrostatics of the interaction of the biomolecules. This relationship can be applied more widely to polyelectrolyte complexes; for example, it has been used to interpret the salt dependence of the neomycin class of drug binding to rRNA [8]. This relationship (Equation 4) was established through studies at relatively low salt concentrations, using spectroscopic techniques to measure KB. Those studies found a consistent fall in binding affinity with increasing salt concentration — an observation that is in agreement the expectations of both the original polyelectrolyte theory and idealised Poisson–Boltzmann models [9]. Recent ITC studies of the binding of the TATA-binding protein (TBP) from the halophilic organism Pyrococcus woesei (Pw) to DNA have shown the wide range of applicability of Equation 4, but suggest a reassessment of the physical interpretation of the equation (Figure 1). PwTBP has increasing affinity for cognate DNA with www.sciencedirect.com
increasing salt concentration [10]. This was explained by assuming that parameter A in Equation 4 could be negative, that is, by assuming that ions could be taken up in the complex. In this case, the equivalent of two cations is taken up by the complex. This interpretation has subsequently been vindicated through a series of simple charge-changing amino acid mutations, which convert PwTBP to the opposite behaviour — weaker binding at high salt [11]. The physical picture that emerges from these experiments is that the binding of PwTBP to the DNA creates, through its charge disposition and a dielectric focusing effect, a diffuse region surrounding the interface that has an enhanced affinity for cations above that of the isolated DNA. The apparent uptake of two cations in the wild-type complex should be interpreted as the aggregate partial occupancy of many spatially diffuse, thermodynamically weak binding sites. This ITC study also showed that binding becomes increasingly entropically favourable with increasing salt to 1 M, regardless of whether or not ions are taken up or released, and that the major difference in the thermodynamics of salt dependence between the wild-type and mutant proteins is enthalpic. This is difficult to reconcile with the conclusions of several theoretical models [7,12], which suggest that the entropy of counterion release (ion release becomes less favourable if there are more ions in solution) accounts for the majority of the salt dependence of protein–DNA interactions. Interesting data on dihydrofolate reductases from another halophile, Haloferax volcanii, also pose a test for theoretical modelling. These enzymes have increased affinity for dihydrofolate, but reduced affinity for the NADPH cofactor, with increased salt [13].
Hydration and heat capacity changes upon binding Changes in heat capacity upon formation of a complex can provide information on non-local effects. Although most interactions will provide some change in heat capacity, it seems that hydrogen-bonded networks of water molecules are particularly sensitive to environmental changes upon formation of a complex. Consequently, a correlation exists between the change in heat capacity of an aqueous system and the extent of hydration of non-polar and polar molecular surfaces. Changes in the solvent-exposed surface area of biomolecules upon binding cause a net change in heat capacity that, in its best-known form, is summarised in the following empirical relationship: DCp ¼ 1:34DAnp 0:59DAp
(5)
where DCp is in units of J.mol1.K1, and DAnp and DAp are the change in non-polar and polar accessible surface area (A˚ 2), respectively [14]. Several versions of the correlation have been fine-tuned using different data sets or Current Opinion in Structural Biology 2004, 14:562–569
564 Biophysical methods
Figure 1
have been tailored to particular types of systems [15,16,17]. Many binding events fit such relationships reasonably well. As a result, when the experimentally determined DCp does not match that calculated from the structure of the complex, it is often assumed that some additional process is occurring. Indeed, such a discrepancy is often the main evidence put forward for coupled folding and binding. However, several recent studies of the heat capacity effects of series of site-directed mutations imply that Equation (5) and similar relationships have limited applicability and fail to adequately encompass the underlying physical processes.
(a) 2.5
Log [KB × 10–5]
2
1.5
1
Water release dominant
Ion binding/release dominant
0.5
–1.25
–1
–0.75
–0.5
–0.25
0
0.25
Log [salt] (b)
Wild type
Mutant
Sites of mutation
Current Opinion in Structural Biology
Reversal of the charge dependence of DNA binding by PwTBP by site-directed mutation [11]. (a) The log(KB) of binding DNA is plotted against log(salt concentration) for the wild-type protein (open squares) and the charge-changing quadruple mutant E12A/E41K/E42K/E128A (filled diamonds). Below a salt concentration of 1M, the effects of ion binding or release are dominant in Equation 4. Uptake of cations upon binding of the wild-type protein leads to a more favourable binding affinity for DNA with increasing salt concentration. The mutations reduce the affinity of the complex for cations, leading to net release of ions upon binding and decreasing affinity for DNA with increasing salt concentration. (b) Possible locations of counterions on the surface of the PwTBP–DNA complex, coloured by electrostatic potential (red is negative potential and thus favourable to cation binding). Note that favourable binding locations are continuous on the DNA surface and, consequently, there are no substantial barriers to cation movement. Also, because binding of cations will neutralise the
Current Opinion in Structural Biology 2004, 14:562–569
Recent studies of the impact of the mutation of biomolecules on the heat capacity of folding [18], protein– protein association [19,20] and protein–DNA interactions [21] have all found changes in heat capacity resulting from point mutations that are an order of magnitude greater than those predicted from empirical relationships. The large changes in DCp associated with individual mutations mean that the difference between the observed DCp and that calculated from empirical relationships, such as Equation 5, cannot be taken as definite evidence of coupled folding and binding. It seems that small local structural changes may be propagated to a much larger region via disruption or creation of a cooperative network of interactions. In the reported example of a protein–DNA interaction, for which there are X-ray structures of both states, the large changes in DCp arise from the mutation of polar residues, in a highly polar and hydrated environment, to apolar residues. The changes in this ‘hyperpolar’ cleft [21] at the border of the interface disrupt a highly organised water-mediated hydrogen-bond network between charged groups on the protein and the phosphate backbone. It is suggested that the local effects of disruption are transmitted to surrounding sidechains and nearby water molecules through rearrangement and weakening of the cooperative network (Figure 2).
Folding coupled to complex formation The DH versus temperature plots for most binding interactions appear to be linear, that is to say, the DCp of binding is constant (see, for example, Figure 2). However, biomolecules are not rigid bodies, but populate and exchange between many structural substates. These substates will have different heat capacities and, because their populations change with temperature, the likely differential changes in heat capacity of the binding partners and the complex will create curvature of the plot of DH versus temperature. Thus, the observed linear appearance is probably a result of limitations of the accuracy of instrumentation and experimental procedures, complex, only a few of these sites will be occupied at any one time. The mutations substantially diminish the size of regions that are favourable to cation binding. The molecular surface of the DNA is shown in white, the protein backbone in green and the mutated residues are represented by pale blue stick models. www.sciencedirect.com
Measuring non-local effects in biomolecular interactions Ladbury and Williams 565
Figure 2
(a)
(b) 120
∆H (kJ/mol)
100
Q103 E12
80
60
25
30
35
40
45
50
55
Temperature (ºC) Current Opinion in Structural Biology
Particular site-directed mutations have unexpectedly large effects on the thermodynamics of a hydrated ‘hyperpolar’ cleft bordering a binding interface. (a) The enthalpy change upon PwTBP binding DNA versus temperature is shown for the wild-type protein (open squares), a Q103A mutant (filled triangle) and a Q103A/E12A double mutant (diamonds). The polar to apolar mutations produce additive reductions of the magnitude of the slope of this plot. (b) E12 and Q103 lie near the centres of two symmetry-related and evolutionarily conserved hydrated clefts (structures of TBP from four species — Pw, human, Arabidopsis thaliana and Saccharomyces cerevisiae — are superimposed), in which the protein sidechains interact indirectly with the phosphate backbone of the DNA via a water-mediated hydrogen-bond network (blue spheres represent water). It is hypothesized that the disruption of these cooperative networks, which probably include further surface water molecules not identifiable by crystallography, by the mutations to alanine leads to the huge effect on heat capacity.
and the small temperature range over which binding events are usually investigated. It is only in a few fortunate cases and with exceptionally diligent experimentation [22,23] that curvature can currently be measured for ‘ordinary’ binding events. However, when a protein folds upon binding its ligand (perhaps the ultimate ‘extended interface’), the substantial change in biomolecular surface hydration may give rise to a much more readily observable curvature. The large heat capacity difference between folded and unfolded protein means that, provided the midpoint of the folding event is close to the experimental temperature range [24], the proportion of folded and unfolded protein contributing to the observed DH at any temperature will vary significantly. If the folding event can be characterised in isolation, then it is possible to deconvolute the DH of folding from the total DH at any temperature [25,26], based on the observed total enthalpy change (DHtot) at any temperature being the sum of components from the binding and the folding: DHtot ¼ DHbind þ aDHfold
(6)
where a is the fraction of unfolded biomolecule at the specified temperature.
Measuring ‘solvent release’ upon binding The measurement of changes in volumetric quantities upon complexation, including partial molar volume, comwww.sciencedirect.com
pressibility and expansivity, provides novel insight into extensive effects of the binding event upon its environment [27]. Changes arise through the impact of the binding event on the intrinsic compressibility of the protein itself, through reorientation and repacking of sidechains and larger scale changes in conformation, plus the change in the influence of the biomolecules on the surrounding solvent. A new framework has been proposed for deconvolving these effects on experimental observations of folding and binding [28]. This has been used in a bold joint volumetric and calorimetric study of the binding of ovomucoid third domain to a-chymotrypsin, in which the number of waters released upon binding are first determined via the change in partial molar volume and then their thermodynamic contribution is estimated on a ‘per water molecule’ basis [29]. A related aspect of the non-local effect of (or influence upon) binding is the effect of molecular crowding, whereby the behaviour of hydrating water is modified by the presence of other solutes. This phenomenon has been recently reviewed in this journal [30] and we only note that an important new interpretative model [31] has been published, which seeks to reconcile the observations of hydration changes due to osmotic stress and pressure, and rationalise the observation of osmolyte-dependent effects [32–34] in such studies. Current Opinion in Structural Biology 2004, 14:562–569
566 Biophysical methods
An intriguing point in many volumetric studies is the persistent observation that many more solvent molecules are ‘released’ than could have occupied a monolayer covering the buried surface ([29] and references therein). This implies either that there is a mistake in the interpretative model or that the biological macromolecule’s influence extends two or three solvent layers into the bulk solution. Support for the latter interpretation comes from neutron scattering data [35] and IR spectroscopic studies of hydration of hydrophobic surfaces [36]. Acceptance of such an extensive impact of the protein on the hydrating water would necessitate revision of many earlier quantitative estimates of hydration thermodynamics based on the monolayer model. Pressure perturbation calorimetry (PPC) is a novel variation of differential scanning calorimetry (DSC) that allows the differential heating effect of an applied pressure jump on reference and sample cells to be measured [37,38]. Because the heat corresponds to the work done by/against pressure to create a volume change, this method obtains the expansivity of the protein partial specific volume, which is strongly correlated with hydration effects. Recent improvements in DSC instruments mean that the sensitivity of this method is an order of magnitude [39] greater than current densitometry and ultrasonic techniques for measuring this quantity, and consequently this new technique should have considerable impact on investigating hydration effects. An early application has already placed a major question mark over ‘solvent-structuring’ models of osmolyte effects [40].
Structural and dynamic change upon binding NMR is the only tool available for complete structural characterisation in solution at atomic resolution and, as such, is the essential complement to thermodynamic studies. The chemical shift (frequency) of each nucleus is determined by the manner in which its local electronic environment shields or enhances the magnetic field of the spectrometer. Upon binding a ligand, the region of the protein that undergoes the greatest change in its local electronic environment, and hence shows greatest changes in chemical shift, is often the binding site. However, such experiments also frequently reveal changes that occur at a distance from the binding site of the ligand (Figure 3). Because shielding is short range (5 A˚ or less), the changes in these shifts show that the effects of binding are propagated through the protein from residue to residue via changes in hydrogen-bond strength or patterns, and sidechain and/or backbone reorientation. The chemical shift changes induced upon binding a ligand consequently provide an exquisitely detailed map of the location of changes in the average electronic environment of the protein’s atoms. As the chemical shifts are quantum mechanical in nature, it may be beyond our ability to calculate them at present; Current Opinion in Structural Biology 2004, 14:562–569
Figure 3
The extensive effects of the localised binding of phosphotyrosine to the v-src SH2 domain, as revealed by NMR (JE Ladbury et al., unpublished). (a) Backbone of the SH2 domain coloured by the change in proton chemical shift of each residue’s backbone amide in going from the apo form to the bound state. The largest chemical shifts are found in the crystallographically identified binding pocket (the crystallographic phosphotyrosine position is shown). However, the chemical shift data also reveal changes in the average local structure throughout the protein that accompany the binding event. (b) Change in the backbone dynamics of each residue, as represented by the change in the Lipari–Szabo order parameter (see [41] for an explanation), for each amide group upon binding. Regions in red show greater extent of movement and regions in blue smaller extent of movement in the bound form.
however, they remain an important challenge for future computation. In addition to monitoring changes in average structure via chemical shift, NMR can also measure the extent and time-scale of local motions by a variety of means [41,42]. In particular, the relaxation of an NMR signal back to its equilibrium value is strongly dependent on the local motion of bond vectors. Measurement of the differential relaxation behaviour of apo and ligand-bound protein reveals that changes in dynamics are also widespread. Of particular importance is the notion that changes in local dynamics can be related to changes in local entropy [43–45]. The idea being that, because bond vector motion on a model potential surface has calculable entropy and a characteristic physical extent, measurement of the extent of the motion by NMR can be used to estimate entropy. Estimates of heat capacity can also be made by studying the temperature dependence of these dynamics [46,47]. Clearly, such local insight would be invaluable in deconvolving conformational effects within the total changes in entropy and heat capacity of binding. The precise quantitative interpretation of the relaxation data in terms of thermodynamics is still not resolved. Several idealised models of the energy surface for N–H bond vector motion and potential surfaces extracted from molecular dynamics simulations [48] have been suggested. However, the qualitative features of the effects www.sciencedirect.com
Measuring non-local effects in biomolecular interactions Ladbury and Williams 567
of ligand binding are fascinating in themselves. Recent studies of the mouse major urinary protein, by two different groups [49,50,51,52], have shown that backbone motions throughout the protein are increased upon binding. Sidechain motions that are proximal to the ligand decrease, but sidechain motions at a greater distance from the binding site increase, in an apparently compensatory manner. Both groups have also performed ITC studies of the system to try to deconvolve motional effects from the experimental total DS. In this respect, the situation is much improved since early studies, which tried to correlate such observations with free energy changes (for which there was no clear thermodynamic justification). Bearing in mind the uncertainty in calculating heat capacities, these studies also imply that there is little heat capacity change arising from backbone or sidechain dynamics, a conclusion that is further supported by an extensive study of a calmodulin–peptide complex [53,54]. Thus, it is implicit that heat capacity effects are dominated by solvent and local vibrations.
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Conclusions Calorimetric, volumetric and NMR observations are revealing, in quantitative detail, that the energetic contributions to binding affinity are not simply localised to the direct interactions between the molecules, but contain substantial contributions from structural and dynamic changes that are propagated throughout the protein, and from counterions and hydrating water molecules in the vicinity of the binding site. The current dominant paradigm in structure-energetics, based on burial of surface area, seems to be inadequate to quantitate the extensive effects of binding or the contextdependent effects of subtle local changes that are introduced via mutation or in the binding of series of related inhibitors [55]. We need to move toward the ab initio prediction of the properties of biomolecules from physical principles by using subtle variation to ask hard questions experimentally about the relationship of structural change, both local and non-local, to the thermodynamics of binding, and toward use and development of more realistic computational models of complexes and their environment at the atomistic level. Enthalpyentropy compensation in complex systems of weak interactions means that DG is not always the most revealing thermodynamic measure [56]. Experimentally, DH and DCp are accurately measurable quantities with a far greater range of variation and closer association with particular phenomena, and are apparently exquisitely sensitive to structural detail; computational modelling should also focus on reproducing them.
Acknowledgements We thank Jon Taylor and Radwan Fawaz for use of their unpublished data in Figure 3. The authors’ work in this area is supported by a Wellcome Trust Senior Research Fellowship (JEL), BBSRC grant 31/C11098, and BBSRC, MRC and UCL Graduate School PhD Studentships. www.sciencedirect.com
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45. Yang DW, Kay LE: Contributions to conformational entropy arising from bond vector fluctuations measured from NMRderived order parameters: Application to protein folding. J Mol Biol 1996, 263:369-382.
28. Chalikian TV, Filfil R: How large are the volume changes accompanying protein transitions and binding? Biophys Chem 2003, 104:489-499. 29. Filfil R, Chalikian TV: The thermodynamics of protein-protein recognition as characterized by a combination of volumetric and calorimetric techniques: The binding of turkey ovomucoid third domain to alpha-chymotrypsin. J Mol Biol 2003, 326:1271-1288. A bold use of combined volumetric and calorimetric measurements in an attempt to deconvolve the contribution of hydration effects to binding. However, the long chain of reasoning from the data to the final estimates (in which the definitions of thermal volume, intrinsic volume and a selection of data from model systems to aid in interpretation are all arguable) leaves considerable room for doubt about the conclusions. 30. Minton AP: Implications of macromolecular crowding for protein assembly. Curr Opin Struct Biol 2000, 10:34-39. 31. Shimizu S: Estimating hydration changes upon biomolecular reactions from osmotic stress, high pressure, and preferential hydration experiments. Proc Natl Acad Sci USA 2004, 101:1195-1199. Shimizu introduces a new theoretical framework for interpreting osmotic and volumetric measurements, which may help to resolve previous discrepancies between the two techniques. 32. Courtenay ES, Capp MW, Anderson CF, Record MT: Vapor pressure osmometry studies of osmolyte-protein interactions: Current Opinion in Structural Biology 2004, 14:562–569
46. Mandel AM, Akke M, Palmer AG: Dynamics of ribonuclease H: Temperature dependence of dynamics on multiple time scales. Biochemistry 1996, 35:16009-16023. 47. Yang DW, Mok YK, Forman-Kay JD, Farrow NA, Kay LE: Contributions to protein entropy and heat capacity from bond vector motions measured by NMR spin relaxation. J Mol Biol 1997, 272:790-804. 48. Massi F, Palmer AG: Temperature dependence of NMR order parameters and protein dynamics. J Am Chem Soc 2003, 125:11158-11159. 49. Zidek L, Novotny MV, Stone MJ: Increased backbone conformational entropy upon hydrophobic ligand binding. Nat Struct Biol 1999, 6:1118-1121. 50. Bingham RJ, Findlay JBC, Hsieh SY, Kalverda AP, Kjeliberg A, Perazzolo C, Phillips SEV, Seshadri K, Trinh CH, Turnbull WB et al.: Thermodynamics of binding of 2-methoxy-3isopropylpyrazine and 2-methoxy-3-isobutylpyrazine to the major urinary protein. J Am Chem Soc 2004, 126:1675-1681. A combined use of NMR and calorimetry to dissect the entropic contributions to binding. The results imply a conformational relay of dynamic changes that is substantially altered by small changes in the ligand. www.sciencedirect.com
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51. Sharrow SD, Novotny MV, Stone MJ: Thermodynamic analysis of binding between mouse major urinary protein-I and the pheromone 2-sec-butyl-4,5-dihydrothiazole. Biochemistry 2003, 42:6302-6309. 52. Krizova H, Zidek L, Stone MJ, Novotny MV, Sklenar V: Temperature-dependent spectral density analysis applied to monitoring backbone dynamics of major urinary protein-I complexed with the pheromone 2-sec-butyl-4,5dihydrothiazole. J Biomol NMR 2004, 28:369-384. 53. Lee AL, Sharp KA, Kranz JK, Song XJ, Wand AJ: Temperature dependence of the internal dynamics of a calmodulin-peptide complex. Biochemistry 2002, 41:13814-13825. 54. Prabhu NV, Lee AL, Wand AJ, Sharp KA: Dynamics and entropy of a calmodulin-peptide complex studied by NMR and molecular dynamics. Biochemistry 2003, 42:562-570.
55. Talhout R, Villa A, Mark AE, Engberts JBFFN: Understanding binding affinity: A combined isothermal titration calorimetry/molecular dynamics study of the binding of a series of hydrophobically modified benzamidinium chloride inhibitors to trypsin. J Am Chem Soc 2003, 125:10570-10579. A combination of advanced modelling and calorimetry, revealing the inadequacies of simpler surface-area-based models. Unfortunately, the more advanced atomistic simulations are not substantially better correlated with experiment. 56. Cooper A, Johnson CM, Lakey JH, Nollmann M: Heat does not come in different colours: entropy-enthalpy compensation, free energy windows, quantum confinement, pressure perturbation calorimetry, solvation and the multiple causes of heat capacity effects in biomolecular interactions. Biophys Chem 2001, 93:215-230.
Free journals for developing countries The WHO and six medical journal publishers have launched the Access to Research Initiative, which enables nearly 70 of the world’s poorest countries to gain free access to biomedical literature through the Internet. The science publishers, Blackwell, Elsevier, the Harcourt Worldwide STM group, Wolters Kluwer International Health and Science, Springer-Verlag and John Wiley, were approached by the WHO and the British Medical Journal in 2001. Initially, more than 1000 journals will be available for free or at significantly reduced prices to universities, medical schools, research and public institutions in developing countries. The second stage involves extending this initiative to institutions in other countries. Gro Harlem Brundtland, director-general for the WHO, said that this initiative was ‘perhaps the biggest step ever taken towards reducing the health information gap between rich and poor countries’. See http://www.healthinternetwork.net for more information.
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Current Opinion in Structural Biology 2004, 14:562–569
and entropy (DS) resulting from the binding process [1]. However, the specific attribution of thermodynamic parameters to the formation/breaking of particular local non-covalent interactions, to conformational or dynamic change, or to solvent reorganisation is not easy to achieve. Even knowledge of the crystal structures of both the free biomolecules and the complex is not enough to allow us to explain the binding thermodynamics. The true extent of conformational change and dynamics can be hidden by crystal packing effects, and mobile parts of the structure, water molecules and salt, are usually indistinguishable or undetectable in smeared or low electron density. Consequently, for a complete understanding of the binding event, we need extensive solution-state experimentation. In this review, we highlight instruments and methodologies that can be employed to deconvolve the non-local structural and thermodynamic effects associated with binding, and show that their application is leading to a fundamental change in the way we think about biomolecular interactions.
Current Opinion in Structural Biology 2004, 14:562–569 This review comes from a themed issue on Biophysical methods Edited by Arthur G Palmer III and Randy J Read Available online 15th September 2004 0959-440X/$ – see front matter # 2004 Elsevier Ltd. All rights reserved. DOI 10.1016/j.sbi.2004.08.001
Abbreviations ITC isothermal titration calorimetry PwTBP Pyrococcus woesei TATA-binding protein
Introduction The idea that biomolecular interactions can be understood simply in terms of the coming together of two rigid bodies to create several specific interactions at a welldefined binding site is progressively being eroded by detailed studies of equilibrium binding processes in solution. There are an increased number of recent reports of binding events that are accompanied by significant structural and dynamic changes of the interacting partners, or perturbation of the surrounding solvent. Often, these changes are distant from the site of intermolecular contact itself, but their contribution to the thermodynamics of binding can be considerable. Consequently, their characterisation is essential to a complete understanding of biological mechanism. Calorimetric methods can determine the total change in free energy (DG), enthalpy (DH) Current Opinion in Structural Biology 2004, 14:562–569
Global thermodynamics of binding Although it has been possible for many years to study the thermodynamics of a binding event indirectly via spectroscopic observation of the bound fraction during a titration, instrumentation that allows a direct and complete thermodynamic characterisation of biomolecular interactions has only recently become commercially available. Isothermal titration calorimetry (ITC) directly determines the total DH of binding and the equilibrium binding constant, KB, is simultaneously determined by using the measured heat as a probe of the degree of binding as two biomolecules are titrated against one another. A complete thermodynamic characterisation of an interaction can be achieved using the relationships: KB ¼ eDG=RT
(1)
DH ¼ DG þ T DS
(2) 1
1
where R is the gas constant (8.3 J.mol .K ) and T is the experimental temperature. Repetition of the experiment at several temperatures (and constant pressure) yields the change in heat capacity (DCp) that accompanies binding: DCp ¼
@DH @T
(3)
There are already several reviews detailing instrument design, usage and applications of the basic ITC technique [1–3]; here, we focus on methods that give insight into the contribution of the ionic environment, protein conformational change and water molecules to binding www.sciencedirect.com
Measuring non-local effects in biomolecular interactions Ladbury and Williams 563
thermodynamics. In order to deconvolve specific contributions, a series of experiments is performed in which a single physical parameter related to the contribution or contributions of interest is systematically varied.
The counterion cloud The contribution of the ionic environment can be experimentally assessed by thermodynamic characterisation at several distinct salt concentrations. Intracellular salt concentrations vary widely, from approximately 200 mM in human tissue to more than 2 M in some halophilic archaea, and their effects can be profound, particularly for oppositely charged binding partners (e.g. the KB of the purine repressor protein for its operator DNA sequence falls by four orders of magnitude as the salt concentration is changed only twofold from 200 mM to 400 mM [4]). The partial cancellation of the electrostatic fields of the protein and DNA as they form the complex radically reduces the range of their effect on the surrounding solution, and the field close to the interface is extensively modified through the focusing effect of the low dielectric biomolecules [5]. Counterions in solution redistribute in response to these changes in order to minimise the free energy of the solute–solvent system. For protein–DNA complexes, an empirical relationship has been established between the salt concentration and the binding constant (derived from [6]): log KB ¼ log KB;ref Alog½Salt þ 0:016B½Salt
(4)
where the constants A and B are interpreted, respectively, as the number of ions and the number of water molecules released to bulk from the negative polyelectrolyte DNA and largely positive protein surfaces that are occluded upon binding [7]. KB,ref is the hypothetical binding constant at 1 M salt and B = 0. The thermodynamic contribution to the salt dependence from the release of water molecules is small below 1 M salt and is a bulk solution property that is not affected by the electrostatics of the interaction of the biomolecules. This relationship can be applied more widely to polyelectrolyte complexes; for example, it has been used to interpret the salt dependence of the neomycin class of drug binding to rRNA [8]. This relationship (Equation 4) was established through studies at relatively low salt concentrations, using spectroscopic techniques to measure KB. Those studies found a consistent fall in binding affinity with increasing salt concentration — an observation that is in agreement the expectations of both the original polyelectrolyte theory and idealised Poisson–Boltzmann models [9]. Recent ITC studies of the binding of the TATA-binding protein (TBP) from the halophilic organism Pyrococcus woesei (Pw) to DNA have shown the wide range of applicability of Equation 4, but suggest a reassessment of the physical interpretation of the equation (Figure 1). PwTBP has increasing affinity for cognate DNA with www.sciencedirect.com
increasing salt concentration [10]. This was explained by assuming that parameter A in Equation 4 could be negative, that is, by assuming that ions could be taken up in the complex. In this case, the equivalent of two cations is taken up by the complex. This interpretation has subsequently been vindicated through a series of simple charge-changing amino acid mutations, which convert PwTBP to the opposite behaviour — weaker binding at high salt [11]. The physical picture that emerges from these experiments is that the binding of PwTBP to the DNA creates, through its charge disposition and a dielectric focusing effect, a diffuse region surrounding the interface that has an enhanced affinity for cations above that of the isolated DNA. The apparent uptake of two cations in the wild-type complex should be interpreted as the aggregate partial occupancy of many spatially diffuse, thermodynamically weak binding sites. This ITC study also showed that binding becomes increasingly entropically favourable with increasing salt to 1 M, regardless of whether or not ions are taken up or released, and that the major difference in the thermodynamics of salt dependence between the wild-type and mutant proteins is enthalpic. This is difficult to reconcile with the conclusions of several theoretical models [7,12], which suggest that the entropy of counterion release (ion release becomes less favourable if there are more ions in solution) accounts for the majority of the salt dependence of protein–DNA interactions. Interesting data on dihydrofolate reductases from another halophile, Haloferax volcanii, also pose a test for theoretical modelling. These enzymes have increased affinity for dihydrofolate, but reduced affinity for the NADPH cofactor, with increased salt [13].
Hydration and heat capacity changes upon binding Changes in heat capacity upon formation of a complex can provide information on non-local effects. Although most interactions will provide some change in heat capacity, it seems that hydrogen-bonded networks of water molecules are particularly sensitive to environmental changes upon formation of a complex. Consequently, a correlation exists between the change in heat capacity of an aqueous system and the extent of hydration of non-polar and polar molecular surfaces. Changes in the solvent-exposed surface area of biomolecules upon binding cause a net change in heat capacity that, in its best-known form, is summarised in the following empirical relationship: DCp ¼ 1:34DAnp 0:59DAp
(5)
where DCp is in units of J.mol1.K1, and DAnp and DAp are the change in non-polar and polar accessible surface area (A˚ 2), respectively [14]. Several versions of the correlation have been fine-tuned using different data sets or Current Opinion in Structural Biology 2004, 14:562–569
564 Biophysical methods
Figure 1
have been tailored to particular types of systems [15,16,17]. Many binding events fit such relationships reasonably well. As a result, when the experimentally determined DCp does not match that calculated from the structure of the complex, it is often assumed that some additional process is occurring. Indeed, such a discrepancy is often the main evidence put forward for coupled folding and binding. However, several recent studies of the heat capacity effects of series of site-directed mutations imply that Equation (5) and similar relationships have limited applicability and fail to adequately encompass the underlying physical processes.
(a) 2.5
Log [KB × 10–5]
2
1.5
1
Water release dominant
Ion binding/release dominant
0.5
–1.25
–1
–0.75
–0.5
–0.25
0
0.25
Log [salt] (b)
Wild type
Mutant
Sites of mutation
Current Opinion in Structural Biology
Reversal of the charge dependence of DNA binding by PwTBP by site-directed mutation [11]. (a) The log(KB) of binding DNA is plotted against log(salt concentration) for the wild-type protein (open squares) and the charge-changing quadruple mutant E12A/E41K/E42K/E128A (filled diamonds). Below a salt concentration of 1M, the effects of ion binding or release are dominant in Equation 4. Uptake of cations upon binding of the wild-type protein leads to a more favourable binding affinity for DNA with increasing salt concentration. The mutations reduce the affinity of the complex for cations, leading to net release of ions upon binding and decreasing affinity for DNA with increasing salt concentration. (b) Possible locations of counterions on the surface of the PwTBP–DNA complex, coloured by electrostatic potential (red is negative potential and thus favourable to cation binding). Note that favourable binding locations are continuous on the DNA surface and, consequently, there are no substantial barriers to cation movement. Also, because binding of cations will neutralise the
Current Opinion in Structural Biology 2004, 14:562–569
Recent studies of the impact of the mutation of biomolecules on the heat capacity of folding [18], protein– protein association [19,20] and protein–DNA interactions [21] have all found changes in heat capacity resulting from point mutations that are an order of magnitude greater than those predicted from empirical relationships. The large changes in DCp associated with individual mutations mean that the difference between the observed DCp and that calculated from empirical relationships, such as Equation 5, cannot be taken as definite evidence of coupled folding and binding. It seems that small local structural changes may be propagated to a much larger region via disruption or creation of a cooperative network of interactions. In the reported example of a protein–DNA interaction, for which there are X-ray structures of both states, the large changes in DCp arise from the mutation of polar residues, in a highly polar and hydrated environment, to apolar residues. The changes in this ‘hyperpolar’ cleft [21] at the border of the interface disrupt a highly organised water-mediated hydrogen-bond network between charged groups on the protein and the phosphate backbone. It is suggested that the local effects of disruption are transmitted to surrounding sidechains and nearby water molecules through rearrangement and weakening of the cooperative network (Figure 2).
Folding coupled to complex formation The DH versus temperature plots for most binding interactions appear to be linear, that is to say, the DCp of binding is constant (see, for example, Figure 2). However, biomolecules are not rigid bodies, but populate and exchange between many structural substates. These substates will have different heat capacities and, because their populations change with temperature, the likely differential changes in heat capacity of the binding partners and the complex will create curvature of the plot of DH versus temperature. Thus, the observed linear appearance is probably a result of limitations of the accuracy of instrumentation and experimental procedures, complex, only a few of these sites will be occupied at any one time. The mutations substantially diminish the size of regions that are favourable to cation binding. The molecular surface of the DNA is shown in white, the protein backbone in green and the mutated residues are represented by pale blue stick models. www.sciencedirect.com
Measuring non-local effects in biomolecular interactions Ladbury and Williams 565
Figure 2
(a)
(b) 120
∆H (kJ/mol)
100
Q103 E12
80
60
25
30
35
40
45
50
55
Temperature (ºC) Current Opinion in Structural Biology
Particular site-directed mutations have unexpectedly large effects on the thermodynamics of a hydrated ‘hyperpolar’ cleft bordering a binding interface. (a) The enthalpy change upon PwTBP binding DNA versus temperature is shown for the wild-type protein (open squares), a Q103A mutant (filled triangle) and a Q103A/E12A double mutant (diamonds). The polar to apolar mutations produce additive reductions of the magnitude of the slope of this plot. (b) E12 and Q103 lie near the centres of two symmetry-related and evolutionarily conserved hydrated clefts (structures of TBP from four species — Pw, human, Arabidopsis thaliana and Saccharomyces cerevisiae — are superimposed), in which the protein sidechains interact indirectly with the phosphate backbone of the DNA via a water-mediated hydrogen-bond network (blue spheres represent water). It is hypothesized that the disruption of these cooperative networks, which probably include further surface water molecules not identifiable by crystallography, by the mutations to alanine leads to the huge effect on heat capacity.
and the small temperature range over which binding events are usually investigated. It is only in a few fortunate cases and with exceptionally diligent experimentation [22,23] that curvature can currently be measured for ‘ordinary’ binding events. However, when a protein folds upon binding its ligand (perhaps the ultimate ‘extended interface’), the substantial change in biomolecular surface hydration may give rise to a much more readily observable curvature. The large heat capacity difference between folded and unfolded protein means that, provided the midpoint of the folding event is close to the experimental temperature range [24], the proportion of folded and unfolded protein contributing to the observed DH at any temperature will vary significantly. If the folding event can be characterised in isolation, then it is possible to deconvolute the DH of folding from the total DH at any temperature [25,26], based on the observed total enthalpy change (DHtot) at any temperature being the sum of components from the binding and the folding: DHtot ¼ DHbind þ aDHfold
(6)
where a is the fraction of unfolded biomolecule at the specified temperature.
Measuring ‘solvent release’ upon binding The measurement of changes in volumetric quantities upon complexation, including partial molar volume, comwww.sciencedirect.com
pressibility and expansivity, provides novel insight into extensive effects of the binding event upon its environment [27]. Changes arise through the impact of the binding event on the intrinsic compressibility of the protein itself, through reorientation and repacking of sidechains and larger scale changes in conformation, plus the change in the influence of the biomolecules on the surrounding solvent. A new framework has been proposed for deconvolving these effects on experimental observations of folding and binding [28]. This has been used in a bold joint volumetric and calorimetric study of the binding of ovomucoid third domain to a-chymotrypsin, in which the number of waters released upon binding are first determined via the change in partial molar volume and then their thermodynamic contribution is estimated on a ‘per water molecule’ basis [29]. A related aspect of the non-local effect of (or influence upon) binding is the effect of molecular crowding, whereby the behaviour of hydrating water is modified by the presence of other solutes. This phenomenon has been recently reviewed in this journal [30] and we only note that an important new interpretative model [31] has been published, which seeks to reconcile the observations of hydration changes due to osmotic stress and pressure, and rationalise the observation of osmolyte-dependent effects [32–34] in such studies. Current Opinion in Structural Biology 2004, 14:562–569
566 Biophysical methods
An intriguing point in many volumetric studies is the persistent observation that many more solvent molecules are ‘released’ than could have occupied a monolayer covering the buried surface ([29] and references therein). This implies either that there is a mistake in the interpretative model or that the biological macromolecule’s influence extends two or three solvent layers into the bulk solution. Support for the latter interpretation comes from neutron scattering data [35] and IR spectroscopic studies of hydration of hydrophobic surfaces [36]. Acceptance of such an extensive impact of the protein on the hydrating water would necessitate revision of many earlier quantitative estimates of hydration thermodynamics based on the monolayer model. Pressure perturbation calorimetry (PPC) is a novel variation of differential scanning calorimetry (DSC) that allows the differential heating effect of an applied pressure jump on reference and sample cells to be measured [37,38]. Because the heat corresponds to the work done by/against pressure to create a volume change, this method obtains the expansivity of the protein partial specific volume, which is strongly correlated with hydration effects. Recent improvements in DSC instruments mean that the sensitivity of this method is an order of magnitude [39] greater than current densitometry and ultrasonic techniques for measuring this quantity, and consequently this new technique should have considerable impact on investigating hydration effects. An early application has already placed a major question mark over ‘solvent-structuring’ models of osmolyte effects [40].
Structural and dynamic change upon binding NMR is the only tool available for complete structural characterisation in solution at atomic resolution and, as such, is the essential complement to thermodynamic studies. The chemical shift (frequency) of each nucleus is determined by the manner in which its local electronic environment shields or enhances the magnetic field of the spectrometer. Upon binding a ligand, the region of the protein that undergoes the greatest change in its local electronic environment, and hence shows greatest changes in chemical shift, is often the binding site. However, such experiments also frequently reveal changes that occur at a distance from the binding site of the ligand (Figure 3). Because shielding is short range (5 A˚ or less), the changes in these shifts show that the effects of binding are propagated through the protein from residue to residue via changes in hydrogen-bond strength or patterns, and sidechain and/or backbone reorientation. The chemical shift changes induced upon binding a ligand consequently provide an exquisitely detailed map of the location of changes in the average electronic environment of the protein’s atoms. As the chemical shifts are quantum mechanical in nature, it may be beyond our ability to calculate them at present; Current Opinion in Structural Biology 2004, 14:562–569
Figure 3
The extensive effects of the localised binding of phosphotyrosine to the v-src SH2 domain, as revealed by NMR (JE Ladbury et al., unpublished). (a) Backbone of the SH2 domain coloured by the change in proton chemical shift of each residue’s backbone amide in going from the apo form to the bound state. The largest chemical shifts are found in the crystallographically identified binding pocket (the crystallographic phosphotyrosine position is shown). However, the chemical shift data also reveal changes in the average local structure throughout the protein that accompany the binding event. (b) Change in the backbone dynamics of each residue, as represented by the change in the Lipari–Szabo order parameter (see [41] for an explanation), for each amide group upon binding. Regions in red show greater extent of movement and regions in blue smaller extent of movement in the bound form.
however, they remain an important challenge for future computation. In addition to monitoring changes in average structure via chemical shift, NMR can also measure the extent and time-scale of local motions by a variety of means [41,42]. In particular, the relaxation of an NMR signal back to its equilibrium value is strongly dependent on the local motion of bond vectors. Measurement of the differential relaxation behaviour of apo and ligand-bound protein reveals that changes in dynamics are also widespread. Of particular importance is the notion that changes in local dynamics can be related to changes in local entropy [43–45]. The idea being that, because bond vector motion on a model potential surface has calculable entropy and a characteristic physical extent, measurement of the extent of the motion by NMR can be used to estimate entropy. Estimates of heat capacity can also be made by studying the temperature dependence of these dynamics [46,47]. Clearly, such local insight would be invaluable in deconvolving conformational effects within the total changes in entropy and heat capacity of binding. The precise quantitative interpretation of the relaxation data in terms of thermodynamics is still not resolved. Several idealised models of the energy surface for N–H bond vector motion and potential surfaces extracted from molecular dynamics simulations [48] have been suggested. However, the qualitative features of the effects www.sciencedirect.com
Measuring non-local effects in biomolecular interactions Ladbury and Williams 567
of ligand binding are fascinating in themselves. Recent studies of the mouse major urinary protein, by two different groups [49,50,51,52], have shown that backbone motions throughout the protein are increased upon binding. Sidechain motions that are proximal to the ligand decrease, but sidechain motions at a greater distance from the binding site increase, in an apparently compensatory manner. Both groups have also performed ITC studies of the system to try to deconvolve motional effects from the experimental total DS. In this respect, the situation is much improved since early studies, which tried to correlate such observations with free energy changes (for which there was no clear thermodynamic justification). Bearing in mind the uncertainty in calculating heat capacities, these studies also imply that there is little heat capacity change arising from backbone or sidechain dynamics, a conclusion that is further supported by an extensive study of a calmodulin–peptide complex [53,54]. Thus, it is implicit that heat capacity effects are dominated by solvent and local vibrations.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1.
Leavitt S, Freire E: Direct measurement of protein binding energetics by isothermal titration calorimetry. Curr Opin Struct Biol 2001, 11:560-566.
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Ladbury JE, Chowdhry BZ: Sensing the heat: the application of isothermal titration calorimetry to thermodynamic studies of biomolecular interactions. Chem Biol 1996, 3:791-801.
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Cliff MJ, Ladbury JE: A survey of the year 2002 literature on applications of isothermal titration calorimetry. J Mol Recognit 2003, 16:383-391.
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Moraitis MI, Xu H, Matthews KS: Ion concentration and temperature dependence of DNA binding: comparison of PurR and LacI repressor proteins. Biochemistry 2001, 40:8109-8117.
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Elcock AH, McCammon JA: The low dielectric interior of proteins is sufficient to cause major structural changes in DNA on association. J Am Chem Soc 1996, 118:3787-3788.
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Ha JH, Capp MW, Hohenwalter MD, Baskerville M, Record MT: Thermodynamic stoichiometries of participation of water, cations and anions in specific and non-specific-binding of lac repressor to DNA - possible thermodynamic origins of the glutamate effect on protein-DNA interactions. J Mol Biol 1992, 228:252-264.
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Record MT, Anderson CF, Lohman TM: Thermodynamic analysis of ion effects on the binding and conformational equilibria of proteins and nucleic acids: the roles of ion association or release, screening and ion effects on water activity. Q Rev Biophys 1978, 11:103-178.
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Kaul M, Pilch DS: Thermodynamics of aminoglycoside-rRNA recognition: The binding of neomycin-class aminoglycosides to the A site of 16S rRNA. Biochemistry 2002, 41:7695-7706.
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Sharp KA, Friedman RA, Misra V, Hecht J, Honig B: Salt effects on polyelectrolyte-ligand binding - Comparison of Poisson-Boltzmann, and limiting law counterion binding models. Biopolymers 1995, 36:245-262.
Conclusions Calorimetric, volumetric and NMR observations are revealing, in quantitative detail, that the energetic contributions to binding affinity are not simply localised to the direct interactions between the molecules, but contain substantial contributions from structural and dynamic changes that are propagated throughout the protein, and from counterions and hydrating water molecules in the vicinity of the binding site. The current dominant paradigm in structure-energetics, based on burial of surface area, seems to be inadequate to quantitate the extensive effects of binding or the contextdependent effects of subtle local changes that are introduced via mutation or in the binding of series of related inhibitors [55]. We need to move toward the ab initio prediction of the properties of biomolecules from physical principles by using subtle variation to ask hard questions experimentally about the relationship of structural change, both local and non-local, to the thermodynamics of binding, and toward use and development of more realistic computational models of complexes and their environment at the atomistic level. Enthalpyentropy compensation in complex systems of weak interactions means that DG is not always the most revealing thermodynamic measure [56]. Experimentally, DH and DCp are accurately measurable quantities with a far greater range of variation and closer association with particular phenomena, and are apparently exquisitely sensitive to structural detail; computational modelling should also focus on reproducing them.
Acknowledgements We thank Jon Taylor and Radwan Fawaz for use of their unpublished data in Figure 3. The authors’ work in this area is supported by a Wellcome Trust Senior Research Fellowship (JEL), BBSRC grant 31/C11098, and BBSRC, MRC and UCL Graduate School PhD Studentships. www.sciencedirect.com
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Free journals for developing countries The WHO and six medical journal publishers have launched the Access to Research Initiative, which enables nearly 70 of the world’s poorest countries to gain free access to biomedical literature through the Internet. The science publishers, Blackwell, Elsevier, the Harcourt Worldwide STM group, Wolters Kluwer International Health and Science, Springer-Verlag and John Wiley, were approached by the WHO and the British Medical Journal in 2001. Initially, more than 1000 journals will be available for free or at significantly reduced prices to universities, medical schools, research and public institutions in developing countries. The second stage involves extending this initiative to institutions in other countries. Gro Harlem Brundtland, director-general for the WHO, said that this initiative was ‘perhaps the biggest step ever taken towards reducing the health information gap between rich and poor countries’. See http://www.healthinternetwork.net for more information.
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Current Opinion in Structural Biology 2004, 14:562–569